This invention pertains generally to the field of biology and particularly to techniques and apparatus for the analysis and sequencing of DNA and related polymers.
The sequencing of deoxyribonucleic acid (DNA) is a fundamental tool of modern biology and is conventionally carried out in various ways, commonly by processes which separate DNA segments by electrophoresis. See, e.g., Current Protocols In Molecular Biology, Vol. 1, Chapter 7, xe2x80x9cDNA Sequencing,xe2x80x9d 1995. The sequencing of several important genomes has already been completed (e.g., yeast, E. coli), and work is proceeding on the sequencing of other genomes of medical and agricultural importance (e.g., human, C. elegans, Arabidopsis). In the medical context, it will be necessary to xe2x80x9cre-sequencexe2x80x9d the genome of large numbers of human individuals to determine which genotypes are associated with which diseases. Such sequencing techniques can be used to determine which genes are active and which inactive either in specific tissues, such as cancers, or more generally in individuals exhibiting genetically influenced diseases. The results of such investigations can allow identification of the proteins that are good targets for new drugs or identification of appropriate genetic alterations that may be effective in genetic therapy. Other applications lie in fields such as soil ecology or pathology where it would be desirable to be able to isolate DNA from any soil or tissue sample and use probes from ribosomal DNA sequences from all known microbes to identify the microbes present in the sample.
The conventional sequencing of DNA using electrophoresis is typically laborious and time consuming. Various alternatives to conventional DNA sequencing have been proposed. One such alternative approach, utilizing an array of oligonucleotide probes synthesized by photolithographic techniques is described in Pease, et al., xe2x80x9cLight-Generated Oligonucleotide Arrays for Rapid DNA Sequence Analysis,xe2x80x9d Proc. Natl. Acad. Sci. USA, Vol. 91, pp. 5022-5026, May 1994. In this approach, the surface of a solid support modified with photolabile protecting groups is illuminated through a photolithographic mask, yielding reactive hydroxyl groups in the illuminated regions. A 3xe2x80x2 activated deoxynucleoside, protected at the 5xe2x80x2 hydroxyl with a photolabile group, is then provided to the surface such that coupling occurs at sites that had been exposed to light. Following capping, and oxidation, the substrate is rinsed and the surface is illuminated through a second mask to expose additional hydroxyl groups for coupling. A second 5xe2x80x2 protected activated deoxynucleoside base is presented to the surface. The selective photodeprotection and coupling cycles are repeated to build up levels of bases until the desired set of probes is obtained. It may be possible to generate high density miniaturized arrays of oligonucleotide probes using such photolithographic techniques wherein the sequence of the oligonucleotide probe at each site in the array is known. These probes can then be used to search for complementary sequences on a target strand of DNA, with detection of the target that has hybridized to particular probes accomplished by the use of fluorescent markers coupled to the targets and inspection by an appropriate fluorescence scanning microscope. A variation of this process using polymeric semiconductor photoresists, which are selectively patterned by photolithographic techniques, rather than using photolabile 5xe2x80x2 protecting groups, is described in McGall, et al., xe2x80x9cLight-Directed Synthesis of High-Density Oligonucleotide Arrays Using Semiconductor Photoresists,xe2x80x9d Proc. Natl. Acad. Sci. USA, Vol. 93, pp. 13555-13560, November 1996, and G. H. McGall, et al., xe2x80x9cThe Efficiency of Light-Directed Synthesis of DNA Arrays on Glass Substrates,xe2x80x9d Journal of the American Chemical Society 119, No. 22, 1997, pp. 5081-5090.
A disadvantage of both of these approaches is that four different lithographic masks are needed for each monomeric base, and the total number of different masks required are thus four times the length of the DNA probe sequences to be synthesized. The high cost of producing the many precision photolithographic masks that are required, and the multiple processing steps required for repositioning of the masks for every exposure, contribute to relatively high costs and lengthy processing times.
In accordance with the present invention, the synthesis of arrays of DNA probe sequences, polypeptides, and the like is carried out rapidly and efficiently using patterning processes. The process may be automated and computer controlled to allow the fabrication of a one or two-dimensional array of probes containing probe sequences customized to a particular investigation. No lithographic masks are required, thus eliminating the significant costs and time delays associated with the production of lithographic masks and avoiding time-consuming manipulation and alignment of multiple masks during the fabrication process of the probe arrays.
In the present invention, a substrate with an active surface to which DNA synthesis linkers have been applied is used to support the probes that are to be fabricated. To activate the active surface of the substrate to provide the first level of bases, a high precision two-dimensional light image is projected onto the substrate, illuminating those pixels in the array on the substrate active surface which are to be activated to bind a first base. The light incident on the pixels in the array to which light is applied deprotects OH groups and makes them available for binding to bases. After this development step, a fluid containing the appropriate base is provided to the active surface of the substrate and the selected base binds to the exposed sites. The process is then repeated to bind another base to a different set of pixel locations, until all of the elements of the two-dimensional array on the substrate surface have an appropriate base bound thereto. The bases bound on the substrate are protected, either with a chemical capable of binding to the bases or with a layer(s) of photoresist covering all of the bound bases, and a new array pattern is then projected and imaged onto the substrate to activate the protecting material in those pixels to which the first new base is to be added. These pixels are then exposed and a solution containing the selected base is applied to the array so that the base binds at the exposed pixel locations. This process is then repeated for all of the other pixel locations in the second level of bases. The process as described may then be repeated for each desired level of bases until the entire selected two-dimensional array of probe sequences has been completed.
The image is projected onto the substrate utilizing an image former having an appropriate light source that provides light to a micromirror device comprising a two-dimensional array of electronically addressable micromirrors, each of which can be selectively tilted between one of at least two separate positions. In one of the positions of each micromirror, the light from the source incident on the micromirror is deflected off an optical axis and away from the substrate, and in a second of the at least two positions of each micromirror, the light is reflected along the optical axis and toward the substrate. Projection optics receive the light reflected from the micromirrors and precisely image the micromirrors onto the active surface of the substrate. Collimating optics may be used to collimate the light from the source into a beam provided directly to the micromirror array or to a beam splitter, wherein the beam splitter reflects a portion of the beam to the micromirror array and transmits reflected light from the micromirror array through the beam splitter. The light directly reflected from the micromirrors or transmitted through the beam splitter is directed to projection optics lenses which image the micromirror array onto the active surface of the substrate. Because the selectively addressable micromirrors in the micromirror array may either fully reflect or fully deflect the light provided to them, the image of the micromirror array exhibits a very high contrast between the xe2x80x9conxe2x80x9d and xe2x80x9coffxe2x80x9d pixels. The micromirrors may also be capable of being indexed to more than two positions, in which case additional optics may be provided to allow exposure of more than one substrate using a single micromirror array device. In addition, the micromirrors are capable of reflecting light at any wavelength without damage to them, allowing short wavelength light, including light in the range of ultraviolet to near ultraviolet light, to be utilized from the light source.
The micromirror array is operated under control of a computer which provides appropriate pixel address signals to the micromirror array to cause the appropriate micromirrors to be in their xe2x80x9creflectxe2x80x9d or xe2x80x9cdeflectxe2x80x9d positions. The appropriate micromirror array pattern for each activation step in each level of bases to be added to the probes is programmed into the computer controller. The computer controller thus controls the sequencing of the images presented by the micromirror array in coordination with the reagents provided to the substrate.
In one embodiment, the substrate may be transparent, allowing the image of the micromirror array to be projected through the surface of the substrate that is opposite to the active surface. The substrate may be mounted within a flow cell, with an enclosure sealing off the active surface of the array, allowing the appropriate reagents to be flowed through the flow cell and over the active surface of the array in the appropriate sequence to build up the probes in the array.